5.6-trans pgb2

ABSTRACT

5,6-Trans-prostaglandins E2, F2 , F2 , A2, and B2 are disclosed. These are useful for the same pharmacological purposes as the known prostaglandins PGE2, PGF2 , PGF2, PGA2, and PGB2.

United States Patent [191 Lincoln, Jr. et al.

54] 5,6-TRANS Pen, I [75] Inventors: Frank H. Lincoln, Jr., Portage;John E. Pike, Kalamazoo, both of Mich.

[73] Assignee: The Upjohn Company, Kalamazoo,

Mich.

[22] Filed: Apr. 23, 1973 [21] App]. No.: 353,481

Related U.S. Application Data [60] Division of Ser. No. 157,651, June28, 1971, Pat. No. 3,759,978, which is a continuation-in-part of Ser.No. 72,51 1, Sept. 15, I970, abandoned.

[52] U.S. Cl. 260/514 D, 260/21 I R, 260/247.2 R, 260/268 R, 260/293.65,260/410, 260/429.9,

260/439 R, 260/448 R, 260/468 D, 260/488 R, 260/501.I, 260/50l.1 S,260/50l.l7, 260/501.2

[11] 3,821,291 June 28, 1974 51 Int. CL, ..C07c 61/36.CQ7C 69/74 [58]Field of Search......... 260/408 D, 514 D, 488 R [5 6] References CitedOTHER PUBLICATIONS Beerthuis et al., Rec. Trav. Chim 90, 943 (1971).

Primary Examiner-Robert Gerstl Attorney, Agent, or Firm-Morris IL.Nielsen [57] ABSTRACT 6 Claims, No Drawings 5,6-TRANS PGBz CROSSREFERENCE TO RELATED APPLICATIONS This is a division of application Ser.No. 157,651, filed June 28, i971 now Pat. No. 3,759,978 which is acontinuation-in-part of our co-pending application Ser. No. 72,51 1,filed Sept. 15, 1970 now abandoned.

DESCRIPTION OF THE INVENTION This invention relates to novelcompositions of matter, to novel methods of producing those, and tonovel chemical intermediates useful in those processes. This inventionis more specifically concerned with novel organic compounds of theformula:

coonI R40 H on, II

or a racemic compound of that formula and the mirror image thereof,wherein R is hydrogen or alkyl of l to 8 carbon atoms, and wherein allRjs are hydrogen, formyl, or alkanoyl of 2 to 8 carbon atoms, inclusive,with the proviso that when R;, is hydrogen or methyl, R, is not hydrogenor acetyl; and the pharmacologically acceptable salts thereof when R;,is hydrogen.

This invention is also concerned with novel organic compounds of theformula:

or a racemic compound of that formula and the mirror image thereof,wherein R is hydrogen or alkyl of l to 8 carbon atoms, wherein all R sare hydrogen, formyl, or alkanoyl of 2 to 8 carbon atoms, inclusive, andwherein indicates attachment of hydroxy to the cyclopentane ring inalpha or beta configuration; and the pharmacologically acceptable saltthereof when R;, is hydrogen.

This invention is also concerned with novel organic compounds of theformula:

wherein R, and R are as defined above, said compound being essentiallyfree of the other components of colonies of the subclass Octocorallia,and the pharmacologically acceptable salts thereof when R, is hydrogen.

This invention is also concerned with novel racemic compounds consistingof Formula IV and the mirror image thereof, wherein R and R are asdefined above; and the pharmacologically acceptable salts thereof whenR, is hydrogen.

This invention is also concerned with novel organic compounds of theformula:

n on. v

or a racemic compound of that formula and the mirror image thereof,wherein R is hydrogen or alkyl of 1' to 8 carbon atoms, and wherein R,is hydrogen, formyl, or alkanoyl of 2 to 8 carbon atoms, inclusive, withthe proviso that when R is hydrogen or methyl, R is not hydrogen oracetyl; and the pharmacologically acceptable salts thereof when R ishydrogen.

. This invention is also concerned with novel organic compounds of theformula:

0 DOOR;

a. H on. v1

or a racemic compound of that formula and the mirror image thereof,wherein R is hydrogen or alkyl of l to 8 carbon atoms, and wherein R, ishydrogen, formyl, or alkanoyl of 2 to 8 carbon atoms, inclusive; and thepharmacologically acceptable salts thereof when R, is hydrogen.

This invention is also concerned with the 15,8 epimers of the aboveFormula-l-to-Vl compounds, i.e. wherein the OH, --OR or -OR groups onC15 are in the beta configuration instead of the alpha (natural)configuration, as will be further discussed below.

These novel compounds of Formulas I to V1, inclusive, and their 15,8epimers are derivatives of prosnumbering: g

VII

Various derivatives of prostanoic acid are known in the art. These arecalled prostaglandins. See, for example, Bergstrom et al., Pharmacol.Rev. 20, l (1968), and references cited therein. For example,prostaglandin E, (PGEQ has the following structure:

| coon Prostaglandin F (PGF has the following structure:

X Prostaglandin F (PGF has the following structure:

XI Prostaglandin A (PHA has the following structure: m I O .7 ll

XII

XIII

ln Formulas Vlll to Xlll, broken line attachments to the cyclopentanering indicate substituents in alpha configuration, i.e., below the planeof the cyclopentane ring. Heavy solid line attachments to thecyclopentane ring indicate substituents in beta configuration, i.e.,above the plane of the cyclopentane ring. The sidechain hydroxy at (1-15in Formulas Vlll to XIII is in S configuration. See Nature, 212, 38(1966) for discusn f t e epqh m strx 9 h flp ost aansi ns-tProstaglandins F F A and B corresponding to PHE, are also known.

It will be observed that the difference between a P0 and a PG is thepresence in the latter of a cis carboncarbon double bond between C-5 andC-6 (see Formula I).

Molecules of the known prostaglandins each have several centers ofasymmetry, and can exist in racemic optically active) form and in eitherof the two enantiomcric (optically active) forms, i.e., thedextrorotatory and levorotatory forms- As drawn, Formulas Vlll to Xllleach represent the particular optically active form of the prostaglandinwhich is obtained from certain mammalian tissues, for example, sheepvesicular glands, swine lung, or human seminal plasma, or by carbonyland/or double bond reduction of a prostaglandin so obtained. See, forexample, Bergstrom et al., cited above.

Each of the novel prostanoic acid derivatives of this invention isencompassed by one of the following formulas:

XIV

XVI

H XVII \WVV R o 4 H XVIII W R o 4 H 0R4 XIX In Formulas XIV to XXI, R ishydrogen or alkyl of l to 8 carbon atoms, inclusive, and R is hydrogen,formyl, or alkanoyl of 2 to 8 carbon atoms, inclusive. In Formulas XIV,XV, XVIII, and XIX, all R15 are the same, i.e. all are hydrogen or allare formyl of alkanoyl. In Formulas XV and XIX, indicates attachment ofhydroxy to the cyclopentane ring in alpha or beta configuration.

In Formulas XIV to XXI and in the formulas recited hereinafter in thespecification and claims, broken line attachments to the cyclopentanering indicate substituents in alpha configuration, i.e., below the planeof the cyclopentane ring. Heavy solid line attachments to thecyclopentane ring indicate substituents in beta configuration, i.e.,above the plane of the cyclopentane ring.

XXI

The side chain hydroxy at C15 in Formulas XIV to XVII is in S (alpha)configuration. That configuration is shown by attachment of saidside-chain hydroxy to 015 with a dotted line and hydrogen with a heavysolid line. The alternative configuration for the side-chain hydroxy atC-l shown in Formulas XVIII to XXI is known as R or epi (beta) and isshown when necessary by attachment of said sidechain hydroxy to C with aheavy solid line and hydrogen with a dotted line, thus The prostaglandincorresponding to 5,6-Trans-PGE (Formula XIV wherein R and R arehydrogen) but with the R or epi configuration at C-l5 will be designated5,6-trans-15B-PGE These conventions regarding formulas, names, andsymbols for derivatives of prostanoic acid apply to the formulas, names,and symbols given hereinafter in the specification and claims.Whenreference is made hereinafter to the compounds of Formulas XIV toXVII, by the symbols 5,6-trans-PGE -PGF -PGF -PGA or -PGB or to theesters or alkanoates of any of those, 15(8) configuration will beintended and by established custom, S or alpha will not be mentioned inthe name or symbol. For all of the other compounds recited hereinafter,the configuration at C-l5 will be identified as 153 whenever the 15(R)configuration is intended.

A significant characteristic of all of the known prostaglandins with acarbon-carbon double bond in the carboxyl-terminated side chain, i.e.,the PG compounds, is cisconfiguration for that double bond.Prostaglandins obtained from animal tissue always have that double bondin cis-configuration. In strikingcon trast, each of the novel prostanoicacid derivatives of this invention has a trans carbon-carbon doublebondbetween C5 and C-6. Since these novel Formula XIV- transderivatives. An alternate method is to name them.

as trans-5,6-dehydro derivatives of the corresponding PG, compoundslThus, when R, and R, in Formulas XIV, XV, XVI, and XVII are hydrogen,the compound defined by Formula XIV is named 5,6-trans-PGE al- 6ternately trans-5,6-dehydro-PGE,. The compounds dcfined by Formula XVare named 5,6-trans-PGF and 5,6trans-PG F according to the configurationof the hydroxy shown in Formula XV as attached tothe ring with a wavyline. The compound defined by Formula XVI is named 5 ,6-trans-PGA andthe compound defined by Formula XVII is named 5,6-transPGB- The 15,8epimers are named similarly, i.e. when R, and R in Formulas XVIII, XIX,and XXI are hydrogen, the compounds are respectively 5,6-trans-l5,B-PGE5,6-trans-l5B-PGF (or -PGF 5,6-trans-l5fi- PGA and 5,6-trans-l5,G-PGB Asin the case of Formulas VIII to XIII. Formulas XIV to XVII are eachintended to represent only optically active prostanoic acid derivativeswiththe same absolute configuration as PGE obtained from mammaliantissues. The racemic or dl" (optically inactive) compounds are thenrepresented as a mixture of that formula and the mirror image thereof.

Formulas XVIII to XXI represent only optically active prostanoic acidderivatives with the 153 IS-epi or 15(R)) configuration. Here also, theracemic compounds of these 158 compounds are then represented as amixture of that formula and the mirror image thereof. 1

PGE PGF PGF PGA and P68 and their esters and pharrnacologicallyacceptable salts, are extremely potent in causing various biologicalresponses. For that reason, these compounds are useful forpharmacological purposes. See, for example, Bergstrom et al.,Pharrnacol. Rev. 20, 1 (1968), and references cited therein. A few ofthose biological responses a systemic arterial blood pressure loweringin the case of the PGE and PGE compounds as measured, for example, inanesthetized (pentobarbital sodium) pentoliniumtreated rats withindwelling aortic and right heart cannulas; pressor activity, similarlymeasured, for the PG F acompounds; stimulation of smooth muscle asshown, for example, by tests on strips of guinea pig ileum, rabbitduodenum, or gerbil colon; potentiation of other smooth musclestimulants; antilipolytic activity as shown by antagonism ofepinephrine-induced mobilization of free fatty acids or inhibition ofthe spontaneous release of glycerol from isolated rat fat pads;inhibition of gastric secretion in the case of the PGE and PGA compoundsas shown in dogs with secretion stimulated. by food orhistamineinfusion; activity on the central nervous system; controllingspasm and facilitating breathing in asthmatic conditions; decreasingblood platelet adhesiveness as shown by platelet-to-glass adhesiveness,and inhibition of blood. platelet aggregation and thrombus formationinduced by various physical stimuli, e.g., arterial injury, and variousbiochemical stimuli, e.g, ADP, ATP, serotonin thrombin, and collagen;and in the case of the PGE and PGB compounds, stimulation of epidermalproliferation and keratini zation as shown when applied in culture toembryonic chick and rat skin segments.

Because of these biological responses, theseknown prostaglandins areuseful to study, prevent, control, or alleviate a wide variety ofdiseases and undesirable. physiological conditions in birds and mammals,including humans, useful domestic animals, pets, and zoologiabout 10mgper m1. of a pharmacologically suitable liquid vehicle or as an aerosolspray, both for topical application.

The PGE, PGFa and PGA compounds are useful in the treatment of asthma.For example, these compounds are useful as bronchodilators or asinhibitors of mediators, such as SRS-A, and histamine which are releasedfrom cells activated by an antigen-antibody complex. Thus, thesecompounds control spasm and facilitate breathing in conditions such asbronchial asthma, bronchitis, bronchiectasis, pneumonia and emphysema.For these purposes, these compounds are administered in a variety ofdosage forms, e.g., orally in the form of tablets, capsules, or liquids;rectally in the form of suppositories; parenterally, subcutaneously, ofintramuscularly, with intravenous administration being preferred inemergency situations; by inhalation in the form or aerosols or solutionsfor nebulizers; or by insufflation in the form of powder. Doses in therange of about 0.01 to 5 mg. per kg. of body weight are used 1 to 4times a day, the exact dose depending on the age, weight, and conditionof the patient and on the frequency and route of administration. For theabove use these prostaglandins can be combined advantageously with otheranti-asthmatic agents, such as sympathomimetics (isoproterenol,phenylephrine, ephedrine, etc); xanthine derivatives (theophylline andaminophyllin); and corticosteriods (ACTH and predinisolone). Regardinguse of these compounds see South African Pat. No. 681,055.

The PGE and PGA compounds are useful in mammals, including man andcertain useful animals, e.g., dogs and pigs, to reduce and controlexcessive gastric secretion, thereby reducing or avoidinggastrointestinal ulcer formation, and accelerating the healing of suchulcers already present in the gastrointestinal tract. For this purpose,the compounds are injected or infused intravenously, subcutaneously, orintramuscularly in an infusion dose range about 0.01 ug. to about 500ug. per kg. of body weight per minute, or in a total daily dose byinjection or infusion in the range about 0.1 to about mg. per kg. ofbody weight per day, the exact dose depending on the age, weight, andcondition of the patient or animal, and on the frequency and route ofadministration.

The PGE PGF and PGF compounds are useful whenever it is desired toinhibit platelet aggregation, to reduce the adhesive character ofplatelets, and to remove or prevent the formation of thrombi in mammals,including man, rabbits, and rats. For example, these compounds areuseful in the treatment and prevention of myocardial infarcts, to treatand prevent post-operative thrombosis, to promote patency of vasculargrafts following surgery, and to treat conditions such asatherosclerosis, arteriosclerosis, blood clotting defects due tolipemia, and other clinical conditions in which the underlying etiologyis associated with lipid imbalance or hyperlipidemia. For thesepurposes, these compounds are administered systemically, e.g.,intravenously, subcutaneously, intramuscularly, and in the form ofsterile implants for prolonged action. For rapid response, especially inemergency situations, the intravenous route of administration ispreferred. Doses in the range about 0.004 to about 20 mg. per kg. ofbody weight per day are used, the exact dose depending on the age,weight, and condition of the patient or animal,

and on the frequency and route of administration.

PGE PGF a and PGF compounds are especially useful as additives to blood,blood products,

blood substitutes, and other fluids which are used in artificialextracorporeal circulation and perfusion of isolated body portions,e.g., limbs and organs, whether attached to the original body, detachedand being preserved or prepared for transplant, or attached to a newbody. During these circulations and perfusions, aggregated plateletstend to block the blood vessels and portions of the circulationapparatus. This blocking is avoided by the presence of these compounds.For this purpose, the compound is added gradually or in single ormultiple portions to the circulating'blood, to the blood of the donoranimal, to the perfused body portion, attached or detached, to therecipient, or to two or all of those at a total steady state dose ofabout 0.001 to 10 mg. per liter of circulating fluid. It is especiallyuseful to use these compounds in laboratory animals, e.g., cats, dogs,rabbits, monkeys, and rats, for these purposes in order to develop newmethods and tech niques for organ and limb transplants.

The PGE compounds are extremely potent in causing stimulation of smoothmuscle, and are also highly active in potentiating other known smoothmuscle stimulators, for example, oxytocic agents, e.g., oxytocin, andthe various ergot alkaloids including derivatives and analogs thereof.Therefore PGE for example, is useful in place of or in combination withless then usual amounts of these known smooth muscle stimulators, forexample, to relieve the symptoms of paralytic ileus, or to control orprevent atonic uterine bleeding after abortion or delivery, to aid inexpulsion of the placenta, and during the puerperium. For the latterpurpose, the PGE compound is administered by intravenous infusionimmediately after abortion or delivery at a dose in the range about 0.01to about 50 pg. per kg. of body weight per minute until the desiredeffect is obtained. Subsequent doses are given by intravenous,subcutaneous, or intramuscular injection or infusion during puerperiumin the range 0.01 to 2 mg. per kg. of body weight per day, the exactdose depending on the age, weight, and condition of the patient oranimal.

The PGE and PGF 3 compounds are useful as hypotensive agents to reduceblood pressure in mammals, including man. For this purpose, thecompounds are administered by intravenous infusion at the rate about0.01 to about 50 ,ug. per kg. of body weight per minute or in single ormultiple doses of about 25 to 500 pg. per kg. of body weight total perday.

The PGE PGA and PGF compounds also increase the burns, intravenous, flowof blood in the mammalian kidney, thereby increasing volume andelectrolyte content of the urine. Therefore, these compounds are usefulin managing cases of renal disfunction, especially those involvingblockage of the renal vascular bed. lllustratively, the compounds areuseful to alleviate and correct cases of edema resulting, for example,from massive surface burns and in the management of shock. For thesepurposes, the compounds are preferably first administered by intravenousinjection at a dose in the range 10 to 1000 pg. per kg of body weight orby intravenous infusion at a dose in the range 0.1 to 20 ug. per kg. ofbody weight per minute until the desired effect is obtained. Subsequentdoses are given by intravensous, intramuscular, or subcutaneousinjection or infusion in the range 0.05 to 2 mg. per kg. of body weightper day.

The PGE PGF and PGF compounds are useful in place of oxytocin to inducelabor in pregnant female animals, including man, cows, sheep, and pigs,at or near term, or in pregnant animals with intrauterpost-mature andnatural labor has not started, or 12 to 60 hours after the membraneshave ruptured and natural labor has not yet started.

The PGF PGF p PGE and PGA compounds are useful for controlling thereproductive cycle in ovulating female mammals, including humans andanimals such as monkeys, rats, rabbits, dogs, cattle, and the like. Forthat purpose, PGF for example, is administered systemically at a doselevel in the range 0.01 mg. to about 20 mg. per kg. of body weight ofthe female mammal, advantageously during a span of time startingapproximately at the time of ovulation and ending approximately at thetime of menses or just prior to menses. Additionally, expulsion of anembryo or a fetus is accomplished by similar administration of thecompound during the first third of the normal mammalian gestationperiod.

As mentioned above, the PGE compounds are potent antagonists ofepinephrine-induced mobilization of free fatty acids. For this reason,this compound is useful in experimental medicine for both in vitro andin vivo studies in mammals, including man, rabbits, and rats, intendedto lead to the understanding, prevention, symptom alleviation, and cureof diseases involving abnormal lipid mobilization and high free fattyacid levels, e.g., diabetes mellitus, vascular diseases, andhyperthyroidism.

The PGE and PGB compounds promote and accelerate the growth of epidermalcells and keratin in animals, including humans, useful domestic animals,pets, zoological specimens, and laboratory animals. For that reason,these compounds are useful to promote and accelerate healing of skinwhich has been damaged, for example, by burns, wounds, and abrasions,and after surgery. These compounds are also useful to promote andaccelerate adherence and growth of skin autografts, especially small,deep (Davis) grafts which are intended to cover skinless areas bysubsequent outward growth rather than initially, and to retard rejectionof homografts.

For these purposes, these compounds are preferably administeredtopically at or near the site where cell growth and keratin formation isdesired, advantageously as an aerosol liquid or micronized powder spray,as an isotonic aqueous solution in the case of wet dressings, or as alotion, cream, or ointment in combination with the usualpharmaceutically acceptable diluents. In some instances, for example,when there is substantial fluid loss as in the case of extensive burnsor skin loss due to other causes, systemic administration isadvantageous, for example, by intravenous injection of infusion,separate or in combination with the usual infusions of blood, plasma, orsubstitutes thereof. Alternative routes of administration aresubcutaneous or intramuscular near the site, oral, sublingual, buccal,rectal, or vaginal. The exact dose depends on such factors as the routeof administration, and the age, weight, and condition of the subject. Toillustrate, a wet dressing for topical application to second and/orthird degree burns of skin'area to 25 square centimeters wouldadvantageously involve use of an isotonic aque ous solution containing 1to 500 ag/ml. of the P68 compound or several times that concentration ofthe PGE compound. Especially for topical use, these prostaglandins areuseful in combination with antibiotics, for example, gentamycin,neomycin, polymyxin B, bacitracin, specinomycin, and oxytetracycline,with other antibacterials, for example, mafenide hydrochloride,sulfadiazine, furazolium chloride, and nitrofurazone, and with corticoidsteroids, for example, hydrocortisone, prednisolone, methylprednisolone,and fluprednisolone, each of those being used in the combination at theusual concentration suitable for its use alone.

The novel 5,6-trans-PG compounds encompassed by Formulas XIV to XVII,and their 153 epimers of Formulas XVlII to XXI each cause the samebiological re sponses described above for the corresponding knownprostaglandins. Each of these new compounds is accordingly useful forthe above described pharmacolog ical purposes, and is used for thosepurposes as described above. 7 V

The known PGE PGF PGF PGA and PGB compounds are all potent in causingmultiple biological responses even at low doses. For example, PG E isextremely potent in causing vasodepression and smooth musclestimulation, andalso are potent as an tilipolytic agents. Moreover, formany applications,

these known prostaglandins have an inconveniently short duration ofbiological activity. ln striking contrast, the novel Formula XlV-to-XXIcompounds are substantially more specific with regard to potency incausing prostaglandine-like biological responses, and have asubstantially longer duration of biological activity. Therefore, each ofthese novel prostaglandin analogs is surprisingly and unexpectedly moreuseful than one of the corresponding above-mentioned known'prostaglandins for at least one of the pharmacological purposesindicated above for the latter, because it has a different and narrowerspectrum of biological activity than the known prostaglandins, andtherefore is more specific in its activity and causes smaller and fewerundesired side effects than when the known prostaglandin is used for thesame purpose. Moreover, because of its prolonged activity, fewer andsmaller doses of the novel prostaglandin analog can frequently be usedto attain the desired result.

The novel Formula XlV-to-XXl compounds are used as described above infree acid form, in alkyl ester form, in formate or alkanoate form, or inpharmacologically acceptable salt form. When the ester form is used, anyalkyl ester can be used wherein the alkyl moiety contains 1 to 8 carbonatoms, inclusive, i.e., methyl, ethyl, propyl, butyl, pentyl, hexyl,heptyl, octyl, and isomeric forms thereof. However, it is preferred thatthe ester be alkyl of one to four carbon atoms, inclusive. Of thosealkyl, methyl and ethyl are especially pre ferred for optimum absorptionof the compound by the body or experimental animal system.

When the formate or alkanoate form is used, all of the hydroxy moietiesare transformed to formate or alkanoate moieties, so that no freehydroxyls remain on the compound. For this purpose, hydroxy hydrogen isreplaced by formyl or alkanoyl or 2 to 8 carbon atoms, e.g., OH istransformed to OCOCl-l Examples of alkanoyl moieties are acetyl,propionyl, butyryl, valeryl, hexanoyl, heptanoyl, octanoyl, and branchedchain alkanoyl isomers of those moieties. Especially preferred amongthese alkanoates for the above described purposes are the acetoxycompounds. These formates and alkanoates are used as free acids, asesters, and in salt fonn as described herein.

Pharmacologically acceptable salts of these prostaglandin analogs usefulfor the purposes described above are those with pharmacologicallyacceptable metal cations, ammonium, amine cations, or quaternaryammonium cations.

Especially preferred metal cations are those derived from the alkalimetals, e.g., lithium, sodium and potassium, and from the alkaline earthmetals, e.g., magnesium and calcium,'.although cationic forms of othermetals, e.g., aluminum, zinc, and iron, are within the scope of thisinvention.

Pharmacologically acceptable amine cations are those derived fromprimary, secondary, or tertiary amines. Examples of suitable amines aremethylamine, dimethylamine, trimethylamine, ethylamine, dibutylamine,triisopropylamine, N-methylhexylamine, decylamine, dodecylamine,allylamine, crotylamine, cyclopentylamine, dicyclohexylamine,benzylamine, dibenzylamine, a-phenylethylamine, ,B-phenylethylamine,ethylenediamine, diethylenetriamine, and like aliphatic, cycloaliphatic,and araliphatic amines containing up to and including about 18 carbonatoms, as well as heterocyclic amines, e.g., piperidence, morpholine,pyr rolidine, piperazine, and lower-alkyl derivatives thereof, e. g.,l-methylpiperidine, 4- ethylmorpholine, l-isopropylpyrrolidine, 2-methylpyrrolidine, 1,4-dimethylpiperazine, 2-

methylpiperidine, and the like, as well as amines containingwater-solubilizing or hydrophilic groups, e.g., mono-, di-, andtriethanolamine, ethyldiethanolamine, N-butylethanolamine,Z-amino-l-butanol, 2amino-2- ethyl-1,3-propanediol, 2-amino-2-methyll-propanol, tris( hydroxymethyl )aminomethane, N- phenylethanolamine,N-(p-tert-amylphenyl)diethanolamine, glactamine, N-methylglucamine, N-methylglucosamine, ephedrine, phenylephrine, epinephrine, procaine, andthe like.

Examples of suitable pharmacologically acceptable quaternary ammoniumcations are tetramethylammonium, tetraethylammonium,benzyltrimethylammonium, phenyltriethylammonium, and the like.

As discussed above, the prostaglandin analogs are administered invarious ways for various purposes; e.g., intravenously, intramuscularly,subcutaneously, orally, intravaginally, rectally, buccally,sublingually, topically, and in the form of sterile implants forprolonged action.

For intravenous injection or infusion, sterile aqueous isotonicsolutions are preferred. For that purpose, it is preferred because ofincreased water solubility to use the free acid form or thepharmacologically acceptable salt form. For subcutaneous orintramuscular injection, sterile solutions or suspensions of the acid,salt, or ester form in aqueous or non-aqueous media are used. Tablets,capsules, and liquid preparations such as syrups, elixirs, and simplesolutions, with the usual pharmaceutical carriers are used for oral orsublingual administration. For rectal or vaginal administration,suppositories, tampons, ring devices, and preparations adapted togenerate sprays or foams or to be used for lavage, all prepared as knownin the art, are used. For tissue implants, a sterile tablet or siliconerubber capsule or other object containing or impregnated with thesubstance is used.

The novel 5,6-trans-PG compounds of Formulas XIV to XVII,- wherein R isas defined above and R, is

12 In Chart A, the initial starting materials are PGE and its alkylesters (Formula XXII), and PGF a and PGF and their alkyl esters (FormulaXXIII). Those acids and alkyl esters, their l5/3-epimers, and racemicmixtures are CHART A HO oo0R=, coon H H on no H on XXII xxnr coon COOK;

XXVI

known in the art or are prepared by methods known in the art. See, forexample, British Specification 1,040,544; E. J. Correy et al., J. Am.Chem. Soc. 91, 5675 (1969) and 92, 397 1970); and W. P. Schneider, Chem.Commun. March 19, 1969, pp. 304-305.

The transformation of PGE and its esters (XXII) to 5,6-trans-PGE and itsesters (XXIV), transformation of PGF and its esters (XXIII) to5,6-trans-PGF a and its esters (XXV), and the transformation of PG F andits esters (XXIII) to 5,6-trans-PFG and its esters (XXV) is effected byexposing a solution containing the Formula- XXII or -XXIII reactant anda diaryl sulfide or diaryl disulfide to ultraviolet radiation in therange 3000 to 5000 A.

For this isomerization any diarylsulfide or diaryl disulfide which isdissociated to aranethiyl radicals by this range of ultravioletradiation and which does not contain functional groups which will reactwith the Formula- XXII or -XXIII starting material can be used. A

weight of the Formula-XXII or -XXIII reactant is usually suitable.

Any liquid organic solvent or mixture of solvents which gives ahomogeneous reaction mixture and which does not react with theFonnula-XXII or -XXIII starting material or the aranethiyl radicals canbe used.

A mixture of benzene and methanol is a suitable reaction solvent.

The exposure to ultraviolet radiation is advantageously carried out inthe range 20 to 30C., although higher or lower reaction temperatures areoperable. Exposure is continued until repeated thin layerchromatography,(TLC) shows no increase in the amount of the desired5,6-trans-PGE product. Usually 24 hours at 25 C. is a suitableirradiation time. The TLC is done on silica gel plates impregnated withsilver nitrate, using chloroform:acetic acidzmethanol (80:10:10) as thesolvent system, and visualization by spraying with 50% aqueous sulfuricacid and then charring.

The desired product is separated from the other reaction mixturecomponents by methods known in the art,

for example, evaporation of the reaction mixture, and

chromatography of the residue.

An alternate method for producing 5,6-trans-PGF 5,6-trans-PGF and theiralkyl esters (XXV) comprises ring carbonyl reduction of thecorresponding Formula-XXIV 5,6-trans-PGE acid or alkyl ester. Mixturesof the 5,6-trans-PGF and 5,6-trans-PGF 5 compounds are thereby obtained.

These ring carbonyl reductions are carried out by methods'known in theart for ring carbonyl reductions by known prostanoic acid derivatives.See, for example, Bergstrom et al., Arkiv Kemi, 19,563 (1963), and

Acta Chem. Scand. 16, 969 (.1962), and British Specifi-.

cation No. 1,097,533. Any reducing agent is used which does not reactwith carbon-carbon double bonds or ester groups. Preferred reagents arelithium (tri-tertbutoxy) aluminum hydride, the metal borohydrides,especially sodium, potassium and zinc borohydrides, and the metaltrialkoxy borohydrides, e.g., sodium trimethoxyborohydride. The mixturesof alpha and beta hydroxy reduction products are separated into theindividualalpha and beta isomers by methods known in the art for theseparation of analogous pairs of known isomeric prostanoic acidderivatives. See, for example, Bergstrom et al., cited above, Granstromet al., J. Biol. Chem. 240, 457 (1965), and Green et al., J. LipidResearch, 5, 1 I7 (1964). Especially preferred as separation methods arepartition chromatographic procedures, both normal and reversed phase,preparative thin layer chromatography, and countercurrent distributionprocedures. I

Referring again to Chart A, 5,6-trans-PGA and its 5,6-trans-PGA acid oralkyl ester" (XXVI) with a base. I

These basic dehydrations and double bond migrations are carried out bymethods known in the art for similar reactions of known prostanoic acidderivatives. See, for example, Bergstrom et al., J. Biol. Chem. 238,3555 (I963). The base is any whose aqueous solution has pH greater than10. Preferred bases are the alkali metal hydroxides. A mixture of waterand sufficient of a water-miscible alkanol to give a homogeneousreaction mixture is suitable as a reaction medium. The PGE type or PGAtype compound is maintained in such a reaction medium until no furtherPGB type compound is formed, as shown by the characteristic ultravioletlight absorption near 278 mp. for the PGB type compound.

The transformations of Chart A to produce the 5,6-trans-PG compounds ofFormulas XIV to XVII are also utilized in producing the corresponding15,8 epimers of Formulas XVIII to XXI. For this purpose, theFormula-XXII and -XXIII initial starting materials are replaced by thecorresponding ISB-PGE and ISB-PGF (or-PGF )compounds. There are thenobtained the corresponding 5,6-transl 5B-PGE 5,6-trans-l5B-PGF (or -PGF5,6-trans-15B- PGA and 5,6-trans-l5B-PGB compounds, either as the freeacids or esters.

Likewise, the racemic 5,6-trans P6 compounds and 7 their ISB-epimers areproduced by the same transforalkyl esters (XXVI) are produced by acidicdehydration of 5,6-trans-PGE and its alkyl esters (XXIV).

These acidic dehydrations are carried out by methods known in the artfor acidic dehydrations of known prostanoic acid derivatives. See, forexample, Pike et al., Proc. Nobel Symposium 11, Stockholm (1966),Intel-science Publishers, New York, p. 162 (1967), and BritishSpecification 1,097,533. Alkanoic acids are 2 to 6 carbon atoms,inclusive, especially acetic acid, are preferred acids for this acidicdehydration. Dilute aqueous solutions of mineral acids, e.g.,hydrochloric acid, especially in the presence of a solubilizing diluent,e.g., tetrahydrofuran, are also useful as reagents for this acidicdehydration, although these reagents may cause partial hydrolysis of anester reactant.

Referring again to Chart A, 5,6-trans-PGB and its alkyl ester (XXVII)are produced either by a basic dehydration of the corresponding5,6-trans-PGE acid or alkylester (XXIV) or by contacting thecorresponding mations shown in Chart A, replacing the P6 compounds withthe appropriate racemic P6 or racemic 15B-PG starting materials.

When a Formula XlV-to-XXI 5,6-trans-PG acid or 5,6-trans-l5,B-PG acid (Ris hydrogen), or the corresponding racemic compound, has been prepared,and an alkyl ester is desired, 'esterification is advantageouslyaccomplished by interaction of the acid with the appropriatediazohydrocarbon. For example, when diazomethane is used, the methylesters are produced. Similar use of diazoethane, diazobutane, and1-diazo-2- ethylhexane, for example, gives the ethyl, butyl, and 2-ethylhexyl esters, respectively.

Esterification with diazohydrocarbons is carried out by mixing asolution of the diazohydrocarbon in a suitable inert solvent, preferablydiethyl. ether, with the acid reactant, advantageously in the same or adifferent inert diluent. After the esterification reaction is complete,the solvent is removed by evaporation, and the ester purified if desiredby conventional methods, preferably by chromatography. It is preferredthat contact of the acid reactants with the diazohydrocarbon be nolonger than necessary to effect the desired esterification, preferablyabout 1 to about 10 minutes, to avoid undesired molecular changes.Diazohydrocarbons are known in the art or can be prepared by methodsknown in the art. See, for example, Organic Reactions, John Wiley &Sons, Inc., New York, N.Y., Vol. 8, pp. 389-394 (1954).

An alternative method for esterification of the carboxyl moiety of thePGF-type or PGE-type compounds comprises transformation of the free acidto the corresponding silver salt, followed by interaction of that saltwith an alkyl iodide. Examples of suitable iodides are methyl iodide,ethyl iodide, butyl iodide, isobutyl iodide, tert-butyl iodide, and thelike. The silver salts are prepared by conventionalmethods, for example,by dissolving the acid in cold dilute aqueous ammonia, evaporating theexcess ammonia at reduced pressure,

and then adding the stoichiometric amount of silver nitrate.

The final Formula XIV-to-XXI acids or esters prepared by the processesof this invention are transformed to alkanoates by interaction of theFormula XIV-to-XXI hydroxy compound with a carboxyacylab ing agent,preferably the anhydride of a lower alkanoic acid, i.e., an alkanoicacid of 2 to 8 carbon atoms, inclusive. For example, use of aceticanhydride gives the corresponding diacetate. Similar use of propionicanhydride, isobutyric anhydride, and hexanoic acid anhydride gives thecorresponding carboxyacylates. In the same manner, the racemic form ofthe Formular XIV- to-XXI compounds yields the corresponding racemicproducts.

The carboxyacylation is advantageously carried out by mixing the hydroxycompound and the acid anhydride, preferably in the presence of atertiary amine such as pyridine or triethylamine. A substantial excessof the anhydride is used, preferably about 10 to about 10,000 moles ofanhydride per mole of the hydroxy compound reactant. The excessanhydride serves as a reaction diluent and solvent. An inert organicdiluent, for example, dioxane, can also be added. It is preferred to useenough of the tertiary amine to neutralizethe carboxylic acid producedby the reaction, as well as any free carboxyl groups present in thehydroxy compound reactant.

The carboxyacylation reaction is preferably carried out inthe rangeabout to about 100 C. The necessary reaction time will depend on suchfactors as the reaction temperature, and the nature of the anhydride andtertiary amine reactants. With acetic anhydride, pyridine, and a 25 C.reaction temperature, a 12 to 24- hour reaction time is used.

The carboxylated produce is isolated from the reaction mixture byconventional methods. For example, the excess anhydride is decomposedwith water, and the resulting mixture acidified and then extracted witha solvent such as diethyl ether. The desired carboxyacylate isrecoveredfrom the diethyl ether extract by evaporation. Thecarboxyacylate is then purified by conventional methods.

By this procedure, the Formula-XIV and -XVIII POE-type hydroxy compoundsare transformed to dialkanoates, the Formula-XV and -XIX PGE-typehydroxy compounds are transformed to trialkanoates, and the Formula XVI,XVII, XX, and XXI PGA-type and PGB-type hydroxy compounds aretransformed to monoalkanoates.

When a formate of a Formula XIV-to-XXI acid or ester is desired for oneof the above-described pharmacological purpose, i.e. when R, in FormulasXIV to XXI is formyl, that is prepared by formylation of thecorresponding Formula XIV-to-XXI hydroxy compound. This formylation iscarried out by procedures known in the art, for example by reaction ofthe hydroxy compound with the mixed anhydride of acetic and formic acidsor with formylimidazole. See, for example, Fieser et al., Reagents forOrganic Synthesis, John Wiley & Sons, Inc., pp. 4and 407 1967) andreferences cited therein.

The novel Formula XIV-to-XXI acids (R and R, are

hydrogen) or their racemic forms are transformed to pharmacologicallyacceptable salts by neutralization with appropriate amounts of thecorresponding inorganic or organic base, examples of which correspond tothe cations and amines listed above. These transformations are carriedout by a variety of procedures known in the art to be generally usefulfor the preparation of inorganic, i.e., metal or ammonium, salts, amineacid addition salts, and quaternary ammonium salts. The choice ofprocedure depends in part upon the solubility characteristics of theparticular salt to be prepared. In the case of the inorganic salts, itis usually suitable to dissolve the acid in water containing thestoichiometric amount of a hydroxide, carbonate, or bicarbonatecorresponding to the inorganic salt desired. For example, such use ofsodium hydroxide, sodium carbonate, or sodium bicarbonate gives asolution of the sodium salt of the prostanoic acid derivative.Evaporation of the water or addition of a water-miscible solvent ofmoderate polarity, for example, a lower alkanol or a lower alkanone,gives the solid inorganic salt if that form is desired.

To produce an amine salt, the acid is dissolved in a suitable solvent ofeither moderate or low polarity. Examples of the former are ethanol,acetone, and ethyl acetate. Examples of the latter are diethyl ether andbenzene. At least a stoichiometric amount of the amine corresponding tothe desired cation is then added to that solution. If the resulting saltdoes not precipitate, it is usually obtained in solid form by additionof a miscible diluent of low polarity or by evaporation. If the amine isrelatively volatile, any excess can easily be removed by evaporation. Itis preferred to use stoichiometric amounts of the less volatile amines.

Salts wherein the cation is quaternary ammonium are produced by mixingthe acid with the stoichiometric amount of the corresponding quaternaryammonium hydroxide in water solution, followed by evaporation of thewater. 7

Since our invention of the novel Formula-I and -IV v compounds and theirISB-epimers, i.e. 5,6-trans-PGE 5,6-trans-PGE methyl ester,5,6-trans-PGE diacetate, 5,6-trans-PGE diacetate methyl ester,5,6-trans-PGA 5,6-trans-PGA methyl ester, 5,6-trans-PGA acetate and5,6-trans-PGA acetate methyl ester, and their l5B-epimers, it has beendiscovered that certain other prostaglandins, i.e., the prostaglandinsknown as PGA- PGA methyl ester, and PGA acetate, methyl ester areobtained by extraction of colonies or colony pieces of the marineinvertebrate Plexaura homomalla (Esper), I791, forma S, and separatingthose compounds from the resulting extract. Also, certain corresponding15- epi compounds, namely l5/3-PGA and l5B-PGA acetate, methyl ester,are known in the art (see, for example, Weinheimer et al., TetrahedronLetters, No. 59, 5185 1969)), and are obtained by extraction of coloniesof Plexaura homomalla (Esper), 1792, forma R.

These Plexaura homomalla forms are members of the subclass Octocorallia,or'derQorgonacea, suborder Holaxonia, family Plexauridae, genusPlexaura. See, for example, Bayer, The Shallow-Water Octocorallia of theWest Indian Region, Martinus Nijhoff, The Hague (1961). Colonies ofthese Plexaura homomalla forms are abundant on the ocean reefs in thezone from the low-tide line to about 25 fathoms in the tropical andsubtropical regions of the western part of the Atlantic Ocean, fromBermuda to the reefs of Brazil, including the eastern shore reefs ofFlorida, the Caribbean island and mainland reefs, and the Gulf of Mexicoisland and mainland reefs. These colonies are bush-like or smalltree-like in habit, and are readily identified for collection asPlexaurti homomalla (Esper), 1792, by those of ordinary skill in thisart. Forms R and S are distinguished by the method described inPreparation 1, below.

Although PGA PGA methyl ester, and PGA acetate, methyl ester, or one ortwo of those appear to be the predominate prostaglandin-like componentsof Plexaura homomalla, forma S, together with small amounts of PGEmethyl ester, it has now been found that small amounts of the 5,6transcompounds, viz. 5,6-trans-PGA 5,6-trans-PGA methyl ester, 5,6-trans-PGAacetate, 5,6-trans-PGA acetate methyl ester, 5,6-trans-PGE and5,6-trans-PGE methyl ester are also obtained from this same organism.This is shown, for example, by the experiments described in Preparations24 below and in the paragraphs following those preparations. It has notyet been conclusively shown that 5,6-trans-PGE diacetate or5,6-trans-PGE diacetate methyl ester are present or obtainable from thissame member or another member of the Octocorallia subclass, but sincePGA acetate methyl ester has been obtained from this source, it seemspossible that 5,6-trans-PGE diacetate and 5,6-trans-PGE diacetate methylester will also be present or obtainable from the same source.

When these 5,6-trans-PGA and 5,6-trans-PGE compounds are obtained from amember of the Octocorallia subclass, e.g., Plexaura homomalla (Esper),1792, forma S, and are intended for one or more of the above-describedpharmacological uses, these compounds must, of course, be obtainedessentially free of the other components of colonies of the subclassOctocorallia. By the term essentially free" here is meant a degree offreedom from these other components except, of course, water, such thatthese 5,6-trans-PGA and 5,6-trans-PGE compounds are suitable for theirintended pharmacological uses, including parenteral administration tohumans. When these compounds are essentially free of these othercomponents of the subclass Octocorallia, they are considered to beessentially pure and usable for all of the above-describedpharmacological uses.

In obtaining the above essentially 5,6-trans-PGE compounds, and, ifpossible, essentially pure 5,6-trans-PGE diacetate and 5,6-trans-PGEdiacetate methylester, from colonies of the subclass Octocorallia, it isimportant that these compounds be essentially free, as that term isdefined above, from other prostaglandin-like compounds. For example, the5,6-trans-PGA -type compounds which are likely to be present in the samecolonies have different spectra of biological activity and, if presentas contaminants, are likely to intefere with the above-describedpharmacological uses of the 5,6-trans-PGE methyl ester and 5,6-trans-PGEdiacetate methyl ester. This degree of purity is readily obtained,especially in the case of 5,6- trans-PGA -type contaminants. There aresubstantial differences in polarity among the various prostaglandins.For example, 5,6-trans-PGA-type prostaglandins are less polar than 5,6-trans-PGE-type prostaglandins. Advantage is easily taken of thesedifferences by using silica gel chromatography, either preparative thinlayer or column.

Furthermore, a procedure for separating the various PGA -type or PGE-type compoundswhich may be present in a member of the subclassOctocorallia from the desired 5,6-trans-PGA -type or 5,6-trans-PGE puretype compounds using a silver resinate column is de scribed inPreparation 4, below. The processes for obtaining PGA -type compoundsand PGE methyl ester from Plexaura omomalla (Esper), 1792, forma Sdescribed in Preparations 2 and 3 below, are not part of our invention.Rather, our invention is the novel and useful compounds of Formulasl-Vland XIV to XXl regardless of how those are prepared.

Although l5B-PGA l5B-PGA methyl ester, and ISB-PGA acetate, methylester, appear to be the predominate prostaglandin-like components ofPlexaura homomalla, forma R, it has now been found that small amounts ofthe 5,6-trans-l5B compounds, viz. 5 ,6-transl 5B-PGA 5,6-trans- 151B-PGA methyl ester, 5,6-trans-15B-PGA acetate, 5,6-trans-15B-PGAacetate methyl ester, 5,6-trans-15B-PGE and 5,6-trans-l5B-PGE methylester are also obtained from this same organism. This is shown, forexample, by the experiments described in Preparations 3-4 below. As forthe corresponding 5,6-trans-l5a compounds, it has not yet beenconclusively shown that 5,6-trans-l5B-PGE diacetate or 5,6-transl5B-PGE5diacetate methyl ester are present or obtainable.

When these 5 ,6 trans-1 5B-PGA and 5,6-trans-l5B-PGE compounds areobtained from a member of the Octocorallia subclass, e.g. Plexaurahomomalla (Esper), 1792, forma R, and are intended for one or more ofthe above-described pharmacological uses, these compounds must, ofcourse, be obtained essentially free of the other components of coloniesof the subclass Octocorallia. By the term essentially free here is meanta degree of freedom from these other components except, of course,water, such that these 5,6-trans-l5B-PGA and 5,6-transl SB-PG Ecompounds are suitable for their intended pharmacological uses,including parenteral administration to humans. When these compounds areessentially free of these other components of the subclass Octocorallia,they are considered to be essentially pure and usable for all of theabove-described pharmacological uses.

In obtaining these 5,6-trans-l5,8-PGE compounds essentially free of5,6-transl 5,B-PGA compounds, use

is made of silica gel chromatography. Also, procedures for separatingl5B-PGA -type or l5B-PGE -type compounds from the desired5,6-trans-l5B-PGA -type or 5,6-trans-l5B-PGE -type compounds areavailable, as set forth below in the Preparations.

The invention can be more fully understood by the following examples.

All temperaturesare in degrees centigrade.

Infrared absorption spectra are recorded on a Perkin- Elmer Model 421infrared spectrophotometer. Except when specified otherwise, undiluted(neat) samples are used.

Ultraviolet spectra are recorded on a Cary Model 15 spectrophotometer.

Mass spectra are recorded on an Atlas CH-4 mass spectrometer with a TO-4source (ionization voltage ev).

The collection of chromatographic eluate fractions starts when theeluant front reaches the bottom of the column.

Brine, herein, refers to an aqueous saturated so dium chloride solution.

The A-IX solvent system used in thin layer chromatography (TLC) is madeup from ethyl acetate-acetic acid-2 2 4-trimethylpentane-water(:20:50c100) ac- Preparation 1 cording to M. Hamberg and B. Samuelsson,J. Biol. Chem. 241, 257 (1966).

To distinguish Plexaura homomalla (Esper), 1792 forma R from Plexaurahomomalla (Esper), 1792 forma S a TLC method is used. A specimenapproximately 2 cm. in length is harvested and placed in a small vialwith a small amount of water if necessary to insure it is wet; and keptclosed for 6-24 hrs. About one ml. of methanol is then added and thesample is either shaken for 2 hrs. at about 25 C or is stored for 16-24hrs. at about 10 C. A sample of the liquid (10-21 A) is spotted on a TLCplate. It is preferred to use a fluorescent-treated silica gel plate,e.g. Uniplate Silica Gel GF (Analtech, lnc., Newark, Del.). As referencestandards, spots of PGA and 15,8-PGA are also applied. The plate isdeveloped in the A-lX System (Hamberg and Sammuelsson, J. Biol. Chem.241, 257 1965)). The spots are finally visualized withvanillin-phosphoric acid spray (McAleer, Arch. Biochem. E. Biophys. 66,120 (1957)). Comparison of the unknown with the two reference spots isthen made and the identity of the coral established (forma Scorresponding to PGA forma R to l5B-PGA Preparation 2 PGA from Plexauralzomomalla (Esper), 1792,

forma S.

Colonies of Plexaura hamomalla (Esper), 1792, forma S, collected fromreefs off the north shore of Jamaica, are frozen by contact with solidcarbon dioxide within one hour after removal from the reef waters. Thefrozen colonies are maintained in insulated boxes containing solidcarbon dioxide (temperature below about 20 C.) until ready for thawing.Then, the frozen colonies (700 g.) are ground to a small particle size(Waring blender) and mixed with 1500 ml. of water. The mixture ismaintained about 20 hours at 25 C. with stirring. Then, the mixture isfiltered through a pad of diatomaceous earth, and the filtrate isacidified with concentrated hydrochloric acid to pH about 2-3. Theacidified filtrate is extracted four times with ethyl acetate. Theextracts are combined, filtered, washed with brine, dried with anhydroussodium sulfate, and evaporated under reduced pressure to give 1 l g. ofoily residue.

The solid residue on the diatomaceous earth filter pad is stirred 2hours in methanol (enough to cover said residue) at 25 C. The mixture isthen filtered, and the filtrate is evaporated to give 14 g. of oilyresidue.

The two oily residues are combined and chromatographed on 1500 g. ofacid-washed silica gel, eluting successively with 8 l. of a 25 to 65%gradient of ethyl acetate in Skellysolve B, 8 l. of a 65 to 100%gradient of ethyl acetate in Skellysolve B, and 5 l. of a 2% methanol inethyl acetate, collecting 500-ml. fractions. (Skellysolve B is a mixtureof isomeric hexanes.) Fractions 8-12 are combined and evaporated to givea small amount of PGA containing a trace of PGA methyl ester. Fractions15-18 are combined and evaporated to give 9.54 g. of PGA Preparation 3PGA compounds from Plexaura homomalla (Esper),

1792, forma S.

Colonies of Plexaura homomalla (Esper), 1792, forma S, collected fromreefs off the north shore of Jamaica, are chopped into chunks severalinches long. The chunks are frozen by contact with solid carbon dioxidewithin one hour after removal from the reef waters. The frozen colonypieces are maintained in insulated boxes containing solid carbon dioxide(temperature below about 20 C.) until the time for extraction. Then, thefrozen colony pieces are ground to a small particle size (Mitts andMerrill hogger; average largest dimension about 5. mm). The particles(1500 g.) are then stirred at high speed with 5 gallons ofdichloromethane for 20 minutes at about 25 C. external temperature. Themixture of dichloromethane and particles is then filtered through a padof diatomaceous earth, and the filtrate is evaporated to about a 2-litervolume at 30 C. under reduced pressure. The liquid which remains iswashed with water, dried with sodium sulfate, and evaporated at 30 C.under reduced pressure.

The oily residue (60 g.) is chromatographed on 3 kg. of silica gel wetpacked in Skellysolve B (a mixture of isomeric hexanes), elutingsuccessively with a gradient of 4 l. of Skellysolve B and 4 l. of 20%ethyl acetate in Skellysolve B, 27 l. of 20%, 18 l. of and 8 l. of 75%ethyl acetate in Skellysolve B, collecting 600-ml. fractions. Fractions39-60 are combined and evaporated to give PGA acetate methyl ester.Fractions 74-76 are combined and evaporated to give 1.03 g. of PGA;;methyl ester. Between fractions and 74 those fractions shown by TLC tocontain PGA acetate are combined and evaporated to yield that compound.Likewise, between fractions 60 and 74, those fractions shown by TLC tocontain PGE diacetate methyl ester are combined and evaporated to yieldthat compound. Still later fractions, after fractions 74-76, shown byTLC to contain PGE methyl ester are combinedand evaporated to yield thatcompound.

Detection of the respective compounds by TLC is done by methods known inthe art, e.g. by spotting the extract fractions on a TLC silica gelplate alongside spots of the authentic compounds, developing the platewith the A-IX system, and observing which spots of the extract fractionscorrespond exactly to the spots of the authentic compounds.

Following the procedures of Preparations 2 and 3, but replacing thecolonies of Plexaura homomalla (Esper), 1792, forma S with colonies ofPlexaura homomalla (Esper), 1792, forma R collected from reefs off thesoutheast shore of Florida near Miami, there are obtained thecorresponding 15,8compounds, viz. 15fi-PGA l5B-PGA acetate methyl ester,15,8-PGA methyl ester, ISB-PGA acetate, 15B-PGE diacetate methyl ester,and ISB-PGE methyl ester.

Each of the PGA -type PGE -type, l5B-PGA -type, and ISB-PGE -typecompounds obtained by the procedures of Preparations 2 and 3 and theparagraphs following Preparation 3 contains the corresponding 5,6- transcompound, e.g. the PGA fraction of Preparation 2 contains 5,6-trans-PGASeparation of the 5,6-trans compound from the prostaglandin is done on asilver resinate column as exemplified in Preparation 4. Preparation 45,6-trans-PGA Separation of 5,6-trans-PGA from PGA is done on achromatographic column using a silver-saturated ionexchange resin. Forbackground on such a column see E. A. Emken et al., .1. Am. Oil ChemistsSoc. 41, 388 1964). Preferably a macroreticular ion exchange resin isused, e.g. a sulfonated styrene-divinylbenzene copolymer having surfacearea of 40-50 sq. m./g., 30-40% porosity, and total exchange capacity of4.5-5.0 meg. per gram of dry resin, for example Am- '21 berlyst 15,available from Rohm and Haas Co., Philadelphia, Pa. The acid-form resinis packed in a column, washed with warm water, and converted to thesilver form by passing 10% silver nitrate solution through the columnuntil the effluent shows a pH of 3.5-4.0. The solumn is then washed withwater to re,- move ionic silver, and finally with denatured ethanol(Type 3A). An ethanolic solution of PGA and 5,6-trans-PGA is charged tothe column. Elution with SA alcohol then yields fractions which arecombined according to their content of 5,6-trans-PGA (faster-eluting) orPGA Assay for 5,6-trans-PGA or PGA in the eluate is conveniently done byTLC using silver nitrate-treated silica gel plates (e.g. AnaltechUniplates dipped in saturated ethanolic silver nitrate and dried) anddeveloping with the A-IX system (upper phase from2,2,4-trimethylpentane:ethyl acetatezacetic acid:water in proportions50:90:20: 100). R; ofPGA is 0.45; R; of ,o-trans-PGA is 0.50. Combinedfractions containing 5.6-trans'PGA are concentrated, partioned betweendichloromethane and a little water, dried over sodium sulfate, andconcentrated under reduced pressure to yield the title compound.

Following the procedures of Preparation 4, each of the PGA -type and PGE-type fractions of Preparations 2 and 3 is subjected to a silverresinate column to yield the corresponding 5,6-trans compound, forexample 5,6-trans-PGA methyl ester, 5,6-trans-PGA acetate, 5,6-trans-PGAacetate methyl ester, 5,6-trans-PGE 5 ,6-trans-PGE methyl ester.

Also following the procedures of Preparation 4, each of the ISB-PGA--type and ISB-PGEg-type fractions obtained following the procedures ofPreparation 2 and 3 yields the corresponding 5,6-trans-l5Bcompound.Example 1 5,6-Trans-PGE A solution of PGE (0.50 g.) and diphenyl sulfide(0.50 g.) in a mixture of 100 ml. of benzene and ml. of methanol isplaced in a quartz tube, purged of oxygen by bubbling nitrogen gasslowly through the solution, and then irradiated with 3500 A ultravioletlight (Rayonet photo-chemical reactor) at 25 C. for 24 hours. Theresulting solution is then evaporated under reduced pressure. Theresidue is chromatographed on g. of acid-washed silica gel (SilicarCC-4) eluting successively with 125 ml. of 40% ethyl acetate incyclohexane, 125 ml. of 60% ethyl acetate in cyclohexane, and 125 ml. ofethyl acetate, collecting 25-ml. fractions. The fractions correspondingto the 100% ethyl acetate eluate are combined and evaporated to give 412mg. of residue.

A cation exchange resin (25 g.; Amberlyst-IS) is I washed four timeswith warm water and then packed as an aqueous slurry into a 100-ml.burette tube. The column is washed with 10% aqueous silver nitratesolution until the pH of the effluent is 3.5. The column is washed freeof excess silver ion with water, and then the water in the column iswashed out with ethanol. A solution of the above-mentioned residue (412mg.) in 3 ml. of ethanol is applied to the column, which is then elutedsuccessively with 75 ml. of ethanol and 25 ml. of 10% cyclohexene inethanol at a flow rate of 0.5 ml. per minute. The first ml. of eluate isevaporated to give 206 ml. of 5,6-trans -PGE The next 45 ml. of eluateis evaporated to give 175 mg. of a mixture of 5,6-trans-PGE and PGE Thatmixture is chromatographed on the same column with prior washing of thecolumn with ethanol, eluting as before with ethanol.

22 The first 30 ml. of eluate from this second chromatography isevaporated to give mg. of 5,6-trans-PGE The combined amounts of 5,6-trans-PGE are chromatographed on 20 g. of acid-washed silica gel(Silicar CC-4), eluting successively with 125 ml. each of 20%, 40%, 60%,80%, and ethyl acetate in cyclohexane. The eluates corresponding to the80% and 100% ethyl acetate are combined and evaporated to give 166 mg.of residue. The residue is recrystallized from a mixture of diethylether and Skellys-olve B (a mixture of isomeric hexanes) to give 108 mg.of 5,6-trans-PGE in the form of colorless plates; melting range 7577 C.;infrared spectral absorption at 3340, 3160, 1730, 1710 sh, 1250, 1170,1080, 1075, and 960 cm.

Following the procedure of Example 1, the methyl, ethyl, isopropyl,tert-butyl, and 2-ethylhexyl esters of PGE; are each isomerized to thecorresponding esters of 5 ,6-trans-PGE Following the procedure ofExample 1, but replacing PGE with ISB-PGE there is obtained5,6-trans-l5fl- P615 Likewise replacing PGE with dl-PGE thereis obtaineddl-5,6-trans-PGE Example 2 dl-5,6-Trans-PGE Methyl Ester Following theprocedure of Example 1, the methyl ester of dl-PGE is isomerized to themethyl ester of dl-5,6-trans-PGE (high resolution mass spectrum: M*- 18:348.2284'and peaks at 335, 330, 31,7, and 277).

Following the procedure of Example 1, the ethyl, isopropyl, tert-butyl,and 2-ethylhexyl esters of dl-PGE are each isomerized to thecorresponding esters of dl- 5,6-trans-PGE Example 3 dl-5,6-Trans-l5B-PGEand its methyl ester.

Following the procedure of Example 1, dl-15B-PGE (obtained from thetrichloroethyl ester of dl- 1 5,8-PGE by treatment with Zinc in aceticacid, see W. P. Schneider, Chem. Commun. 304-305, March 19, 1969) isisomerized to dl-5,6-transl 5B-PGE Esterification with diazomethane indiethyl ether, thereafter removing the solvent, yields the methyl esterof dl-5,6-trans- 1 SB-PGE (high resolution mass spectrum: M -18:348.2272 and peaks at 330, 317, 299, and 277).

Alternatively, dl-l5B-PGE is esterified with diazomethane in diethylether, and the resulting methyl ester of dl-15B-PGE is then isomerizedfollowing the procedure of Example 1 to the methyl ester ofdl-5,6-trans-l5,B-PGE having the same properties reported above.

Following the procedure of Example 1, the ethyl, isopropyl, tert-butyl,and 2-ethylhexyl esters of dl-15B-PGE are each isomerized to thecorresponding esters of dl-5,6-trar1sl 5B-PGE Example 4 5,6-Trans-PGFand 5,6-Trans-PGF I I A cold (0 C.) solution of sodium borohydride (200mg. in 10 ml. of methanol is added rapidly to a stirred solution of5,6-trans-PGE (75 mg.) in 5 ml; of methanol at 0 C. The mixture isstirred 30 minutes at 0 C. and then 30 minutes while warming to 25 C.The mixture is evaporated to half its volume, diluted with 10 ml. ofwater, and then evaporated until methanol has been removed. Theresulting mixture is acidified with one N hydrochloric acid and thenextracted 3 times with ethyl acetate. The combined extracts are washedwith water, dried, and evaporated. The residue is chromatographed on 10g. of acid-washed silica gel (Silicar CC-4), eluting successively with50 ml. of 50% ethyl acetate in cyclohexane, 140 ml. of ethyl acetate,and 60 ml. of ethyl acetate containing 1% acetic acid and 2% methanol,collecting IO-ml. fractions. Fractions 9-14 are combined and evaporatedto give 20 mg. of 5,6-trans-PGF melting point 9596 C. after twocrystallizations from diethyl ether. Fractions 1624 are combined andevaporated to give 34 mg. of 5,6-trans-PGF identical on TLC with theproduct of Example 5, below.

Following the procedure of Example 4, the methyl, ethyl, isopropyl,tert-butyl, and 2-ethylhexyl esters of 5,6-trans-PGE are aech reduced tothe corresponding esters of a mixture of 5,6-trans-PGF and 5,6-trans-PGFthe components of each such mixture being separated as described inExample 4.

Thus, from 5,6-trans-PGE methyl ester there is obtained 5,6-trans-PGFmethyl ester, rn.p. 70 C., infrared spectral absorption 'at 3300, 3200,1740, 1320, 1195, 1175, 1150, 1075, 1040, 1020, and 970 cm, massspectral peaks at 368 (M'), 350, 332, and 278.

Following the procedure of Example 4, 5,6-trans-lB-PGE and the methyl,ehtyl, isopropyl, tert-butyl, and Z-ethylhexyl esters of5,6-trans-15B-PGE are each transformed to the corresponding free acidand esters of a mixture of 5,6-trans-15B-PGF and 5,6-trans-l5B-PGF thecomponents of each such mixture being separated by the proceduresdescribed in Example 4.

Following the procedure of Example 4, dl-5,6-trans-PGE and the methyl,ethyl, isopropyl, tert-butyl, and 2-ethylhexyl esters ofdl-5,6-trans-PGE are each reduced to the corresponding free acid andesters of a mixture of dl-5,6-trans-PGF and dl-5,6-trans-PGF thecomponents of each such mixture being separated by the proceduresdescribed in Example 4.

Following the procedure of Example 4, dl-5,6-transl 5/3-PGE and themethyl, ethyl, isopropyl, tert-butyl, and 2-ethylhexyl esters ofdl-5,6-trans-l5B-PGE are each reduced to the corresponding free acid andesters of a mixture of dl-5,6-trans-l5B-PGF and dl-5,6-trans-l5B-PGF thecomponents of each such mixture being separated by the procedures inExample 4.

Example ,5 5,6-Trans-PGF A solution of PGF 1.00 g.) and diphenyl sulfide1.00 g.) in a mixture of methanol ml.) and benzene( 100 ml.) isirradiated for 24 hours at 25 C. as described in Example 1. The reactionmixture is evaporated under reduced pressure, and the residue ischromatographed on 50 g. of acid-washed silica gel (Silicar CC-4),eluting successively with 250 ml. 40% ethyl acetate in cyclohexane, 250ml. 60% ethyl acetate in cyclohexane, 350 ml. ethyl acetate, and 250 ml.of ethyl acetate containing 1% acetic acid and 3% methanol, collecting-50-ml. fractions. Fractions 18-22 corresponding to the last eluent arecombined and evaporated to give 803 mg. of a residue which ischromatographed on a silver-loaded cation exchange resin prepared asdescribed in Example 1 from 50 g. of resin, eluting with ethanol. Thefirst 200 ml. of eluate is evaporated to give 400 mg. of 5,6-trans-PGFThat is chromatographed on 40 g. of acid-washed slica gel (SilicarCC-4), eluting successively with 250 ml. 60% ethyl acetate incyclohexane, 100 ml. 80% ethyl acetate in cyclohexane 500 ml. ethylacetate, and 200 ml. ethyl acetate containing one acetic acid and 2%methanol, collecting 50-ml. fractions. Fractions 11-21 are combined andevaporated to give 316 mg. 5,6-trans-PGF melting range 6869 C. aftercrystallization from diethyl ether and recrystallization from a mixtureof acetone and Skellysolve B; infrared spectral absorption (mineral oilmull) at 3270, 3180 sh, 1710, 1315, 1300, 1255, 1205, 1075, 1065, 1040,1020, 970, and 930 cm".

Following the procedure of Example 5, the methyl, ethyl, isopropyl,tert-butyl, and Z-ethylhexyl esters of PGF are each isomerized to thecorresponding esters of 5,6-trans-PGF each of those being identical withthe ester prepared by carbonyl reduction as described above.

Example 6 5,6-Trans-PGF Following the procedure of Example 5, PGF 175mg.) is isomerized to 5,6-trans-PGF (40 mg.) identical with thatobtained in Example 4.

Also following the procedure of Example 5, the methyl, ethyl, isopropyl,tert-butyl, and 2-ethylhexyl esters of PGF are each isomerized to thecorresponding esters of 5,6-trans-PGF- each of those being identicalwith the ester prepared by carbonyl reduction as described above.-

Example 7 5,6-Trans-PGA A solution of 5,6-trans-PGE (500 mg.) in amixture of glacial acetic acid (9 ml.) and water 1 ml.) is heated undernitrogen at C. for 18 hours. Then, the acetic acid and water areevaporated under reduced pressure, and the residue is chromatographed on50 g. of acidwashed silica gel, eluting with a 25-100% gradient of ethylacetate in Skellysolve B. The fractions containing the desired productfree of starting material as shown by TLC are combined and evaporated togive 5,6-trans. PGA Infrared spectral absorption at 3380, 2640, 1705,1585, 1295, 1240, 1180, 1150, 1015, and 970 cm. High resolution massspectral data for trimethylsilylated compound: M 478.2998.

Following the procedure of Example 7, the methyl, ethyl, isopropyl,tert-butyl, and 2-ethylhexyl esters of 5,6-trans-PGE are eachtransformed to the corresponding ester of 5,6-trans-PGA Following theprocedure of Example 7, 5,6-trans-l5/3-PGE and the methyl, ethyl,isopropyl, tert-butyl, and 2-ethylhexyl esters of 5,6-trans-15B-PGE areeach transformed to the corresponding free acid and esters of5,6-trans-15B-PGA Following the procedure of Example 7, dl-5,6-trans-PGEand the methyl, ethyl, isopropyl, tert-butyl, and 2-ethylhexyl esters ofdl-5,6-trans-PGE are each transformed to the corresponding free acid andesters of dl-5,6-trans-PGA Following the procedure of Example 7,dl-5,6-transl 5B-PGE and the methyl, ethyl, isopropyl, tert-butyl, and2-ethylhexyl esters of dl-5,6-transl SB-PGE are each transformed to thecorresponding free acid and esters of dl-5,6-trans-15B- PGA Example 85,6-Trans-PGB A solution of 5,6-trans-PGE (200 mg.) in ml. of 50%aqueous ethanol containing 1.5 grams of potassium hydroxide is kept at25 C. for 10 hours under ni- V trogen. Then, the solution is cooled to10 C. and neutralized by addition of 3 normal hydrochloric acid at 10 C.The resulting solution is extracted repeatedly with ethyl acetate, andthe combined ethyl acetate extracts are washed with water and then withbrine, dried, and evaporated to give 5,6-trans-PGB Following theprocedure of Example 8, 5,6-trans-PGA is transformed to 5,6-trans-PGBwith properties identical to those of the compound obtained in Example8.

Also following the procedure of Example 8, the methyl, ethyl, isopropyl,tert-butyl, and 2-ethylhexyl esters of 5,6-trans-PGE and 5,6trans-PGAare each transformed to the corresponding ester of 5,6-trans- PGBFollowing the procedure of Example 8, 5,6-trans-l5B-PGE and the methyl,ethyl, isopropyl, tert-butyl, and Z-ethylhexyl esters of5,6-trans-15,8-PGE are each transformed to the corresponding free acidand esters of 5,6-trans-l5,8-PGB Following the procedure of Example 8,dl-5,6-trans-PGE and the methyl, ethyl, isopropyl, tert-butyl, and2-ethylhexyl esters of dl-5,6-trans-PGE are each transformed to thecorresponding free acid and esters of dl-5,6-trans-PGB Following theprocedure of Example 8, dl-5,6-transl 5B-PGE and the methyl, ethyl,isopropyl, tert-butyl, and 2-ethylhexyl esters of dl5,6-trans- B-PGE areeach transformed to the corresponding free acid and esters ofdl-5,6-trans-l5B- PGB f Example 9 5,6-Trans-PGE ll,l5-Diacetate5,6-Trans-PGE (10 mg.) is mixed with acetic anhydride (2 ml.) andpyridine (2 ml.). The resulting mixture is left standing at 25 C. for 18hours. The reaction mixture is then cooled externally with ice, dilutedwith water, and acidified with dilute hydrochloric acid to pH 1. Thatmixture is extracted three times with diethyl ether. The combinedextracts are washed successively with dilute hydrochloric acid, diluteaqueous sodium bicarbonate solution, and water, dried, and evaporated togive the title compound.

Following the procedure of Example 9, but using in place of the freeacid 5,6-trans-PGE compound the methyl, ethyl, isopropyl, tert-butyl,and Z-ethylhexyl esters of 5,6-trans-PGE there are obtained thecorresponding diacetates.

Following the procedure of Example 9, but using propionic anhydride,isobutyric anhydride, and hexanoic anhydride each in place of the aceticanhydride, there are obtained the corresponding dipropionate,diisobutyrate, and dihexanoate of 5,6-tr ans-PGE methyl ester.

Following the procedure of Example 9, but replacing 5 ,6-trans-PGE with5 ,6-trans-l5B-PGE and its methyl, ethyl, isopropyl, tert-butyl, and2-ethylhexyl esters, there are obtained the corresponding diacetates.

Following the procedure of Example 9, dl-5,6-trans-PG E anddl-5,6-trans-l5B-PGE and their methyl, ethyl, isopropyl, tert-butyl and2-ethylhexyl esters are transformed to the corresponding diacetates.

Following the procedure of Example 9, 5,6-trans-PGF 5,6-trans-PGF5,6-trans-l5B-PGF 5,6-trans-l5B-PGF and their methyl, ethyl, isopropyl,tert-butyl, and 2- ethylhexyl esters are transformed to thecorresponding triacetates.

Also following the procedure of Example 9, but replacing 5,6 trans-PGEwith 5,6-trans-PGA 5,6-trans- 15,8-PGA 5,6-trans-PGB 5,6-trans-l5B-PGBand their methyl, ethyl, isopropyl, tert-butyl, and 2 ehtylhexyl estersare transformed to the corresponding monoacetates. Example 10 EsterFollowing the general procedures of Staab et al., Ann. 655, (1962),5,6-trans-PGA methyl ester is transformed to 5,6-trans-PGA formate,methyl ester.

Following the procedure of Example 10, 5,6trans-PGF 5,6-trans-PGF5,6-trans-15B-PGF 5,6-trans-l5,B-PGF and their methyl, ethyl, isopropyl,tert-butyl, and 2- ethylhexyl esters are transformed to thecorresponding triformates.

Following the procedure of Example 10, 5,6-trans PGE 5,6-trans-15B-PGEdl-5,6-trans- PGE dl-5,6-trans-l5B-PGE and their methyl, ethyl,isopropyl, tert-butyl and 2-ethylhexyl esters are transformed to thecorresponding diformates.

Also following the procedure of Example 10, 5,6-trans-PGB is transformedto the corresponding formate.

Example 11 5,6-trans-l5B-PGA Following the procedure of Preparation 4,but using a fraction containing l5B-PGA obtained from Plexaura homomalla(Esper), 1792, forma R, following the procedure of Preparation 2, thereis obtained the 5,6-Trans-PGA Formate, Methyl title compound, havinginfrared spectral absorptions at 3400 broad, 2650, 1725, 1705, 1585,1240, 1175, 1045, 1020, and 970.

We claim:

1. An optically active compound of the formula COORa ac /W or a racemiccompound of that formula and the mirror image thereof, wherein R ishydrogen or alkyl of l to 8 carbon atoms, and wherein R, is hydrogen,formyl, or alkanoyl of 2 to 8 carbon atoms, inclusive, and thepharmacologically acceptable salts thereof when R is hydrogen.

5. 5,6-Trans-l5B-PGB an optically active compound according to claim 4wherein R and R, are hydrogen.

6. dl-5,6-Trans-l5B-PGB a racemic compound according to claim 4 andwherein R and R, are hydrogen. l l l =l UNITED STATES PATENT ANDTRADEMARK OFFICE CETEFECATE OF CORREUHN PATENT N0. 1 3,821,291 DATEDJune 28, 197

INVLNTOWS) Frank H. Li ncol n,Jr. and John E. Pi ke It is certified thaterror appears in the above-identified patent and that said LettersPatent are hereby corrccled as shown below:

Column 3, l i ne 38, PHA should read (PGA l i ne G l,

"PHE should read PGE; Column 4, l i ne 2}, "R C" should read R 0 Column6, line 5 "PGE should read PGF Column 7, l i ne 40, "0.01" should read 1Column 8, l i ne 50, 'i ncrease the burns, i ntravenous, flow" shouldread increase the flow Column 11, line 23, "piperidence' should readpiperidine Column 12, line +8, "PFC-Q 5" should read PGFzE Column 13, li ne 27, by" should read of l i ne 56, "are" should read of Column 15,line 46, "PGE-type" should read PGF-type Column 18, line "omomalla"should read homomal la Column 22, l i ne 24, "Edl should read dl line33, "55a should read dl Signed and Scaled this eighth Day of June 1976SEAL! RUTH C. MASON C. MARSHALL DANN Arresting Officer CommissionerufPamm and Trademark:

2. 5,6-Trans-PGB2, an optically active compound according to claim 1wherein R3 and R4 are hydrogen.
 3. dl-5,6-Trans-PGB2, a racemic compoundaccording to claim 1 wherein R3 and R4 are hydrogen.
 4. An opticallyactive compound of the formula
 5. 5,6-Trans-15 Beta -PGB2, an opticallyactive compound according to claim 4 wherein R3 and R4 are hydrogen. 6.dl-5,6-Trans-15 Beta -PGB2, a racemic compound according to claim 4 andwherein R3 and R4 are hydrogen.